INTRODUCTION

• Electron Microscopes are scientific instruments that use a beam of

highly energetic electrons to examine objects on a very fine scale.

• This examination can yield information about the topography ,

morphology, composition and crystallographic information.

Mainly 2 types:
• Transmission Electron Microscope (TEM) - allows one the
study of the inner structures.

• Scanning Electron Microscope (SEM) - used to visualize the

surface of objects.
:

RESOLVING POWER
Resolving power is the
ability of an imaging
device to see objects
distinctly, that are
located at a small
angular distances.
Close to the eye – At larger distance -not resolvable .
resolvable
Rayleigh's criterion:

Angular resolution = 1.22λ / D

• Smaller value of angular resolution - instrument can resolve finer details

& has a higher resolving power.
• Electrons have very small wavelength.
• Hence according to Rayleigh's criterion, electron wave can be used to
resolve very small angular separations.
 Budding yeast cell

Compound microscope image TEM image SEM image

E. coli bacteria

Compound microscope image TEM image SEM image

TEM image of golgi complex TEM image of mitochondria

SEM image of blood cells Drosophila's eye. SEM image of pollen grains
 PRINCIPLE OF WORKING OF TEM
• Electrons possess a wave like character.

• Electrons emitted into vacuum from a heated

filament with increased accelerating potential
will have small wavelength.

• Such higher-energy electrons can penetrate

distances of several microns into a solid.

• If these transmitted electrons could be focused -

images with much better resolution.

• Focusing relies on the fact that, electrons also

behave as negatively charged particles and are
therefore deflected by electric or magnetic fields.
PARTS OF TEM 1: Electron cannon.

3: Vacuum pumps
system
2. Electro-magnetic
lenses to. direct and
focus
7: Waterthesupply
electron
to beam
4: Opening to insert a grid
cool the column..
the instrument
inside
with samples into the high-
vacuum chamber for
observation..

6: Screen for menu

and image display

5: Operation panels
• The electron source consists of a
cathode and an anode.
• Cathode - tungsten filament which
emits electrons when being heated.
•A negative cap confines the electrons
into a loosely focused beam
• The beam is then accelerated towards
the specimen by the positive anode

Electron beam is tightly focused using

electromagnetic lens and metal
apertures.

A platform equipped with a mechanical

arm for holding the specimen and
controlling its position.

Electromagnetic lens system

Objective lens Projector lens

Phosphorescent Screen
 SAMPLE PREPARATION FOR TEM
• Fixation - fixed with chemical products (e.g. glutaraldehyde)
• Rinsing and ‘staining’ - treated with heavy metal compounds.
• Dehydration - washing with increasing ethanol concentration, followed by
final wash in another a polar substance like propylene oxide.
• Embedding in resin - material is gradually infiltrated with the still
unpolymerized resin .Little pieces of resin-infiltrated material are placed in
small holders.
• Trimming of resin block and ultrathin sectioning - sections with a
thickness of about 70 nm are cut with special knifes of cleaved glass . The
cutting is done with a ultra-microtome.
• Collection of sections on grid
 WORKING OF TEM
• Specimen is bombarded by a beam of electrons, the primary electrons .
The bombarding electrons are focussed to a bundle onto the object.
• In areas in the object where these electrons encounter atoms with a heavy
atomic nucleus, they rebound.
• In regions where the material consists of lighter atoms , the electron are
able to pass through.
• The fine pattern of electrons leaving the object , reaches the objective lens
forms the image
• It is then greatly enlarged by projector lens.
• Eventually, the traversing electrons (transmission) reach the scintillator
plate at the base of the column of the microscope.
• The scintillator contains phosphor compounds that can absorb the energy of
the stricking electrons and convert it to light flashes.
• Thus a contrasted image is formed on this plate.
ADVANTAGES & DISADVANTAGES OF TEM
Advantages
• TEMs offer very powerful magnification and resolution.
• TEMs have a wide-range of applications and can be utilized in a
variety of different scientific, educational and industrial fields
• TEMs provide information on element and compound structure .
• Images are high-quality and detailed.

Disadvantages
• TEMs are large and very expensive.
• Laborious sample preparation.
• Operation and analysis requires special training.
• Samples are limited to those that are electron transparent.
• TEMs require special housing and maintenance.
• Images are black and white .
 BIOLOGICAL APPLICATIONS OF
TEM
• In medicine as a diagnostic tool – important in renal biopsies.
• Cellular tomography
– Tomography refers to imaging by sectioning, through the use of any
kind of penetrating wave.
– Information is collected and used to assemble a three dimensional
image of the target.
– Used for obtaining detailed 3D structures of subcellular
macromolecular objects.
• Cancer research - studies of tumor cell ultrastructure .
• Toxicology – to study the impacts of environmental pollution on the
different levels of biological organization.
 PRINCIPLE OF WORKING OF SEM
• Incoming (primary) electrons
– can be “reflected” (backscattered)
from a bulk specimen.
– can release secondary electrons.
• Primary electrons are focused into a small-
diameter electron probe that is scanned
across the specimen.
• Electrostatic or magnetic fields, applied at
right angles to the beam, can be used to
change its direction of travel.
• By scanning simultaneously in
two perpendicular directions, a square or
rectangular area of specimen (known as a
raster) can be covered.
• Image of this area can be formed by
collecting secondary electrons from each
point on the specimen.
PARTS OF SEM 1.Electron cannon.

6. Screen for menu

2.and
Electro-magnetic
image display
lenses to focus the
3.electron
Vacuumbeam
pumps.
system
.
5. Operation panel with
focus, alignment and
magnification tools and
a 4.Opening
joystick fortopositioning
insert the
7.Cryo-unit
of the
object sample.
into to
theprepare
high-
frozen
vacuummaterial before
observation
insertion in the
chamber.
observation chamber
in Cryo-SEM mode
• Electron gun consisting of
cathode and anode.
• The condenser lens
controls the amount of
electrons travelling down
the column
• The objective lens focuses
the beam into a spot on the
sample.
• Deflection coil helps to
deflect the electron beam.
• SED attracts the secondary
electrons.
• Additional sensors detect
backscattered electrons
and X-rays.
 SEM SAMPLE PREPARATION
•Sample coated with a thin layer of conductive
material.
•Done using a device called a "sputter coater.”
• Sample placed in a small chamber that is at a
vacuum . Sputter coater
•Gold foil is placed in the instrument.
• Argon gas and an electric field cause an
electron to be removed from the argon, making
the atoms positively charged.
• The argon ions then become attracted to a A spider coated in gold
negatively charged gold foil.
•The argon ions knock gold atoms from the
surface of the gold foil.
• These gold atoms fall and settle onto the
surface of the sample producing a thin gold
coating. 13mm radius aluminium stubs
 SEM WORKING
• The electron gun produces an electron beam when tungsten wire is
heated by current.
• This beam is accelerated by the anode.
• The beam travels through electromagnetic fields and lenses, which
focus the beam down toward the sample.
• A mechanism of deflection coils enables to guide the beam so that
it scans the surface of the sample in a rectangular frame.
• When the beam touches the surface of the sample, it produces:
– Secondary electrons (SE)
– Back scattered electrons (BSE)
– X - Rays...
• The emitted SE is collected by SED and convert it into signal that is
sent to a screen which produces final image.
• Additional detectors collect these X-rays, BSE and produce
corresponding images.
• A secondary electron detector attracts the scattered electrons
and, depending on the number of electrons that reach the
detector, registers different levels of brightness on a monitor.

• By reducing the size of the area scanned by the scan coils, the SEM
changes the magnification of the image.
ADVANTAGES & DISADVANTAGES OF SEM
Advantages
• It gives detailed 3D and topographical imaging and the versatile
information garnered from different detectors.
• This instrument works very fast.
• Modern SEMs allow for the generation of data in digital form.
• Most SEM samples require minimal preparation actions.
Disadvantages
• SEMs are expensive and large.
• Special training is required to operate an SEM.
• The preparation of samples can result in artifacts.
• SEMs are limited to solid samples.
• SEMs carry a small risk of radiation exposure associated with the
electrons that scatter from beneath the sample surface.
 BIOLOGICAL APPLICATIONS OF
SEM
• Virology - for investigations of virus structure
• Cryo-electron microscopy – Images can be made of the surface of
frozen materials.
• 3D tissue imaging -
– Helps to know how cells are organized in a 3D network
– Their organization determines how cells can interact.
• Forensics - SEM reveals the presence of materials on evidences that
is otherwise undetectable
• SEM renders detailed 3-D images
– extremely small microorganisms
– anatomical pictures of insect, worm, spore, or other organic
structures
Differences between SEM and TEM
TEM SEM
Electron beam passes through thin Electron beam scans over surface of
sample. sample.

Specially prepared thin samples are Sample can be any thickness and is
supported on TEM grids. mounted on an aluminum stub.

Specimen stage halfway down Specimen stage in the chamber at the

column. bottom of the column.

Image shown on fluorescent screen. Image shown on TV monitor.

Image is a two dimensional Image is of the surface of the sample

projection of the sample.
 RECENT DEVELOPMENTS
• Three famous physicists, Harald H. Rose , Knut W. Urban and
Maximillian Haider have received the Wolf-prize in physics 2011
for the realization of aberration-corrected electron microscopy.
• Aberrations are intrinsic imperfections of electron lenses.
• Those aberrations are reduced by installing in a microscope a set of
specially designed auxiliary "lenses" which are called aberration
correctors.
• They designed a novel aberration corrector thereby improving
resolution of transmission electron microscope.
 CONCLUSION
• Since its invention, electron microscope has been a valuable tool in
the development of scientific theory
• It has contributed greatly to biology, medicine and material
sciences.
• This wide spread use because they permit the observation of
materials on a nanometer (nm) to micrometer (μm) scale.
• Although SEMs and TEMs are large, expensive pieces of
equipments, they remain popular among researchers due to the high-
resolution and detailed images they produce.
 REFERENCES
• Gray, Peter. Encyclopedia of Microscopy and Microtechnique. Van
Nostrand Reinhold Company, New York.
• Narayanan P (2003). Essentials of Biophysics, New Age International
Publishers
• http://www.vcbio.science.ru.nl/en/fesem/info/principe/
• http://www.hkphy.org/atomic_world/tem/tem02_e.html
• http://www.scribd.com/doc/8288963/Physical-Principles-of-Electron-
Microscopy-an-Introduction-to-TEM-SEM-and-AEM#page=26
• http://www.seallabs.com/hiw7.htm
• http://www.youtube.com/watch?v=oKretoPSZ_Q
• http://www.youtube.com/watch?v=aHx7uqyCHwM&feature=related